Biochemical characterization of NfsA, the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp, a Vibrio harveyi flavin oxidoreductase.

We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.     

Conversion of NfsB, a minor Escherichia coli nitroreductase, to a flavin reductase similar in biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri, by a single amino acid substitution.

NfsB is an oxygen-insensitive nitroreductase of Escherichia coli with significant amino acid sequence homology to the major flavin reductase (FRase I) of Vibrio fischeri. Here, we show that NfsB is convertible to an FRase I-like flavin reductase three times as active as the authentic FRase I by a single amino acid substitution in the least-conserved region.     

Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus erythropolis D-1

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K_m values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.     

Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824

The genes that encode oxygen-insensitive nitroreductases from Clostridium acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized. The gene products NitA (MW 31 kDa) and NitB (MW 23 kDa) were purified to homogeneity. The NitA and NitB are oxygen-insensitive nitroreductases comprised of a single nitroreductase domain. NitA and NitB enzymes show spectral characteristics similar to flavoproteins. The biochemical characteristics of NitA and NitB are highly similar to those of NfsA, the major nitroreductase from E. coli. NitA exhibited broad specificity similar to that of E. coli NfsA and displayed no flavin reductase activity. NitB showed broad substrate specificity toward nitrocompounds in a pattern similar to NfsA and NfsB of Escherichia coli. NitB has high sequence similarity to NAD(P)H nitroreductase from Archaeoglobus fulgidus. NitA could utilize only NADH as an electron donor, whereas NitB utilized both NADH and NADPH as electron donors with a preference for NADH. The activity of both nitroreductases was high toward 2,4-Dinitrotoluene (2,4-DNT) as a substrate. Both the nitroreductases were inhibited by dicoumarol and salicyl hydroxamate. The nitroreductases showed higher relative expression on induction with TNT, nitrofurazone and nitrofurantoin compared to the uninduced control.     

Structure and site-directed mutagenesis of a flavoprotein from Escherichia coli that reduces nitrocompounds: alteration of pyridine nucleotide binding by a single amino acid substitution

The crystal structure of a major oxygen-insensitive nitroreductase (NfsA) from Escherichia coli has been solved by the molecular replacement method at 1.7-A resolution. This enzyme is a homodimeric flavoprotein with one FMN cofactor per monomer and catalyzes reduction of nitrocompounds using NADPH. The structure exhibits an alpha + beta-fold, and is comprised of a central domain and an excursion domain. The overall structure of NfsA is similar to the NADPH-dependent flavin reductase of Vibrio harveyi, despite definite difference in the spatial arrangement of residues around the putative substrate-binding site. On the basis of the crystal structure of NfsA and its alignment with the V. harveyi flavin reductase and the NADPH-dependent nitro/flavin reductase of Bacillus subtilis, residues Arg(203) and Arg(208) of the loop region between helices I and J in the vicinity of the catalytic center FMN is predicted as a determinant for NADPH binding. The R203A mutant results in a 33-fold increase in the K(m) value for NADPH indicating that the side chain of Arg(203) plays a key role in binding NADPH possibly to interact with the 2'-phosphate group.     

H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H₂O₂, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H₂O₂ production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The K_m for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H₂O₂ production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H₂O₂ production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H₂O₂ production in L. johnsonii.     

Vibrio harveyi Nitroreductase Is Also a Chromate Reductase

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5α and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5α NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5α NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K_m values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 μM, respectively. The V_max values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V_max/K_m value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30°C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).     

Oxygen-Insensitive Nitroreductases NfsA and NfsB of Escherichia coli Function under Anaerobic Conditions as Lawsone-Dependent Azo Reductases

Quinones can function as redox mediators in the unspecific anaerobic reduction of azo compounds by various bacterial species. These quinones are enzymatically reduced by the bacteria and the resulting hydroquinones then reduce in a purely chemical redox reaction the azo compounds outside of the cells. Recently, it has been demonstrated that the addition of lawsone (2-hydroxy-1,4-naphthoquinone) to anaerobically incubated cells of Escherichia coli resulted in a pronounced increase in the reduction rates of different sulfonated and polymeric azo compounds. In the present study it was attempted to identify the enzyme system(s) responsible for the reduction of lawsone by E. coli and thus for the lawsone-dependent anaerobic azo reductase activity. An NADH-dependent lawsone reductase activity was found in the cytosolic fraction of the cells. The enzyme was purified by column chromatography and the amino-terminal amino acid sequence of the protein was determined. The sequence obtained was identical to the sequence of an oxygen-insensitive nitroreductase (NfsB) described earlier from this organism. Subsequent biochemical tests with the purified lawsone reductase activity confirmed that the lawsone reductase activity detected was identical with NfsB. In addition it was proven that also a second oxygen-insensitive nitroreductase of E. coli (NfsA) is able to reduce lawsone and thus to function under adequate conditions as quinone-dependent azo reductase.     

Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction

Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen-insensitive nitroreductase of Escherichia coli, is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single-electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one-electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni- and di-valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH-catalysed chromate reduction reaction decreased ROS generation by c. 65, 40, or 20%, respectively, suggesting that these enzymes can pre-empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.     

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

A cDNA clone was isolated from a maize (Zea mays L. cv W64A×W183E) scutellum λgt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.     

Binding of the Covalent Flavin Assembly Factor to the Flavoprotein Subunit of Complex II

Escherichia coli harbors two highly conserved homologs of the essential mitochondrial respiratory complex II (succinate:ubiquinone oxidoreductase). Aerobically the bacterium synthesizes succinate:quinone reductase as part of its respiratory chain, whereas under microaerophilic conditions, the quinol:fumarate reductase can be utilized. All complex II enzymes harbor a covalently bound FAD co-factor that is essential for their ability to oxidize succinate. In eukaryotes and many bacteria, assembly of the covalent flavin linkage is facilitated by a small protein assembly factor, termed SdhE in E. coli. How SdhE assists with formation of the covalent flavin bond and how it binds the flavoprotein subunit of complex II remain unknown. Using photo-cross-linking, we report the interaction site between the flavoprotein of complex II and the SdhE assembly factor. These data indicate that SdhE binds to the flavoprotein between two independently folded domains and that this binding mode likely influences the interdomain orientation. In so doing, SdhE likely orients amino acid residues near the dicarboxylate and FAD binding site, which facilitates formation of the covalent flavin linkage. These studies identify how the conserved SdhE assembly factor and its homologs participate in complex II maturation.     

Thermostable Flavin Reductase That Couples with Dibenzothiophene Monooxygenase,from Thermophilic Bacillus sp. DSM411: Purification,Characterization, and Gene Cloning

Flavin reductase is essential for the oxygenases involved in microbial dibenzothiophene (DBT) desulfurization. An enzyme of the thermophilic strain, Bacillus sp. DSM411, was selected to couple with DBT monooxygenase (DszC) from Rhodococcus erythropolis D-1. The flavin reductase was purified to homogeneity from Bacillus sp. DSM411, and the native enzyme was a monomer of M_r 16 kDa. Although the best substrates were flavin mononucleotide and NADH, the enzyme also used other flavin compounds and acted slightly on nitroaromatic compounds and NADPH. The purified enzyme coupled with DszC and had a ferric reductase activity. Among the flavin reductases so far characterized, the present enzyme is the most thermophilic and thermostable. The gene coded for a protein of 155 amino acids with a calculated mass of 17,325 Da. The enzyme was overproduced in Escherichia coli, and the specific activity in the crude extracts was about 440-fold higher than that of the wild-type strain, Bacillus sp. DSM411.     

Ferredoxin-NADP+ Reductase from Pseudomonas putida Functions as a Ferric Reductase

Pseudomonas putida harbors two ferredoxin-NADP⁺ reductases (Fprs) on its chromosome, and their functions remain largely unknown. Ferric reductase is structurally contained within the Fpr superfamily. Interestingly, ferric reductase is not annotated on the chromosome of P. putida. In an effort to elucidate the function of the Fpr as a ferric reductase, we used a variety of biochemical and physiological methods using the wild-type and mutant strains. In both the ferric reductase and flavin reductase assays, FprA and FprB preferentially used NADPH and NADH as electron donors, respectively. Two Fprs prefer a native ferric chelator to a synthetic ferric chelator and utilize free flavin mononucleotide (FMN) as an electron carrier. FprB has a higher k_cat/K_m value for reducing the ferric complex with free FMN. The growth rate of the fprB mutant was reduced more profoundly than that of the fprA mutant, the growth rate of which is also lower than the wild type in ferric iron-containing minimal media. Flavin reductase activity was diminished completely when the cell extracts of the fprB mutant plus NADH were utilized, but not the fprA mutant with NADPH. This indicates that other NADPH-dependent flavin reductases may exist. Interestingly, the structure of the NAD(P) region of FprB, but not of FprA, resembled the ferric reductase (Fre) of Escherichia coli in the homology modeling. This study demonstrates, for the first time, the functions of Fprs in P. putida as flavin and ferric reductases. Furthermore, our results indicated that FprB may perform a crucial role as a NADH-dependent ferric/flavin reductase under iron stress conditions.Commonly, Fprs are ubiquitous, monomeric, reversible flavin enzymes. Fprs evidence a profound preference for NADP(H) over NAD(H) (3). They harbor a prosthetic flavin cofactor (FAD) and catalyze the reversible electron exchange between NADPH and either ferredoxin (Fd) or flavodoxin (Fld) (4, 5). In oxygenic photosynthesis, the Fd is reduced by the photosystem and subsequently passes electrons on to NADP⁺ via the Fpr. This reaction provides the cellular NADPH pool required for CO₂ assimilation and other biosynthetic processes (4, 5). In heterotrophic organisms such as bacteria, reduced ferredoxin, owing to the reverse enzymatic activity of the Fpr, can donate an electron to several Fd-dependent enzymes, such as nitrite reductase, sulfite reductase, glutamate synthase, and Fd-thioredoxin reductase, allowing ferredoxin to function in a variety of systems, including oxidative stress (1, 4, 5).Iron is the fourth most abundant element in the natural environment and exists primarily as an oxidized form, Fe(III), which has very low solubility under neutral pH conditions (9, 34) and thus presents problems in terms of bioavailability. However, ferrous iron, of Fe(II), is soluble and available at neutral pH in bacterial cytosol (34). Most bacteria secrete siderophores, which are natural chelators of ferric iron. After they bind to ferric iron, that complex enters the bacteria and releases ferric iron into the cytosol in ferric or ferrous form (9). In the bacterial cytosol, ferric iron must be reduced to ferrous form, and thus ferric reductase is essential to bacterial iron utilization.Commonly, prokaryotic ferric reductases are divided into two groups—namely, the bacterial and archaeal types (34). The typical bacterial type ferric reductase is Escherichia coli Fre, which also functions as a flavin reductase. In other words, the ferric reductase can reduce free flavin as flavin reductase, rather than having the flavin cofactor as a prosthetic group in E. coli (38). The archaeal ferric reductase harbors a flavin cofactor in the enzyme and thus does not require a flavin carrier for ferric reduction (26, 34). E. coli Fre includes a Rosmann folding structure at the NAD(P) binding region, whereas the archaeal ferric reductase (FeR) of Archaeoglobus fulgidus does not evidence that folding structure (6, 34). Many bacterial ferric reductases utilize free flavins, such as flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and riboflavin, as electron carrier and, NADH (NAD) or NADP as electron donors to ferric reductase (14, 34). However, reduced ferric iron by reduced free flavin gives rise to the Fenton reaction, which generates the hydroxyl radical within the cell (20, 38). The Fenton reaction is known to generate hydroxyl radicals from ferrous iron and hydrogen peroxide (20). The hydroxyl radical is the most reactive radical and can damage DNA, proteins, and membrane lipids (16, 20, 34, 38). Therefore, the fine-tuning of ferric reduction regulation is required for the survival of bacterial cells.Many Pseudomonas strains, including Pseudomonas putida, a gram-negative soil model bacteria, and Pseudomonas aeruginosa, a human pathogen bacteria, do not harbor annotated ferric reductase within their genome sequences. Commonly, the pathogens compete with the host for available iron, whichis crucial for their survival within the host. Thus, studies of P. aeruginosa regarding iron utilization, siderophores, and ferric reduction are considered to be essential for a better understanding of human infections (9, 19). Studying the physiology and ecology of P. putida also provides us with a new framework for elucidating the basis of the metabolic versatility and environmental stress response of soil microorganisms. Thus, the study of ferric reductase in strains of Pseudomonas at the molecular level is certainly required. From the structural perspective, ferric reductases are generally considered to be contained within the structurally diverse ferredoxin-NADP⁺ reductase (Fprs; EC 1.18.1.2) superfamily, which is frequently involved in the transfer of electrons between Fd/Fld and NADP(H) (2, 15, 34). Thus, we tested the role of the Fpr as a ferric reductase using free flavin (FMN or FAD), NADH, or NADPH as electron donors, and ferric-citrate or ferric-EDTA as terminal electron acceptors (37). We determined that FprA could efficiently utilize NADPH in ferric reduction. Rather, FprB could use NADH as an electron donor and may perform a crucial role as a NADH-dependent ferric reductase under iron stress conditions.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3 ^? null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

The Function of Cytoplasmic Flavin Reductases in the Reduction of Azo Dyes by Bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an “azo reductase.” The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic “azo reductases,” which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744.

The gene encoding the major NAD(P)H-flavin oxidoreductase (flavin reductase) of the luminous bacterium Vibrio fischeri ATCC 7744 was isolated by using synthetic oligonucleotide probes corresponding to the N-terminal amino acid sequence of the enzyme. Nucleotide sequence analysis suggested that the major flavin reductase of V. fischeri consisted of 218 amino acids and had a calculated molecular weight of 24,562. Cloned flavin reductase expressed in Escherichia coli was purified virtually to homogeneity, and its basic biochemical properties were examined. As in the major flavin reductase in crude extracts of V. fischeri, cloned flavin reductase showed broad substrate specificity and served well as a catalyst to supply reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. The major flavin reductase of V. fischeri not only showed significant similarity in amino acid sequence to oxygen-insensitive NAD(P)H nitroreductases of Salmonella typhimurium, Enterobacter cloacae, and E. coli but also was associated with a low level of nitroreductase activity. The major flavin reductase of V. fischeri and the nitroreductases of members of the family Enterobacteriaceae would thus appear closely related in evolution and form a novel protein family.     

Residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA

The major O₂-insensitive nitroreductase (NfsA) of Escherichia coli shares low sequence homology but similar biochemical and structural features with NfsB, the E. coli minor O₂-insensitive nitroreductase. A structural comparison revealed Phe42 was present in the active site of NfsA but not NfsB. F42Y, F42N and F42A were generated and had decreased activity toward nitrofurazone by 52, 96, and 99 %, respectively. The kinetic parameters for other nitroaromatic substrates were also determined. Compared to wild type, the mutants did not have significantly altered K _ms, but had dramatically decreased k _cat and k _cat/K _m values. Far-UV CD spectral analysis of the mutants suggested that there were no significant conformational changes however F42A and F42N had changes from 208 to 222 nm, which was attributed to loss of helix content. These findings revealed that Phe42 is important for maintaining NfsA activity and structure.     

NADH-Nitrate Reductase Inhibitor from Soybean Leaves

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.

The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO₃. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V_max.

The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.

Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.

     

Evaluation of NfsA-like nitroreductases from <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> and <Emphasis Type="Italic">Bartonella henselae</Emphasis> for enzyme-prodrug therapy,targeted cellular ablation,and dinitrotoluene bioremediation

Objectives

To characterize the activities of two candidate nitroreductases, Neisseria meningitidis NfsA (NfsA_Nm) and Bartonella henselae (PnbA_Bh), with the nitro-prodrugs, CB1954 and metronidazole, and the environmental pollutants 2,4- and 2,6-dinitrotoluene.

Results

NfsA_Nm and PnbA_Bh were evaluated in Escherichia coli over-expression assays and as His₆-tagged proteins in vitro. With the anti-cancer prodrug CB1954, both enzymes were more effective than the canonical O₂-insensitive nitroreductase E. coli NfsB (NfsB_Ec), NfsA_Nm exhibiting comparable levels of activity to the leading nitroreductase candidate E. coli NfsA (NfsA_Ec). NfsA_Nm is also the first NfsA-family nitroreductase shown to produce a substantial proportion of 4-hydroxylamine end-product. NfsA_Nm and PnbA_Bh were again more efficient than NfsB_Ec at aerobic activation of metronidazole to a cytotoxic form, with NfsA_Nm appearing a promising candidate for improving zebrafish-targeted cell ablation models. NfsA_Nm was also more active than either NfsA_Ec or NfsB_Ec with 2,4- or 2,6-dinitrotoluene substrates, whereas PnbA_Bh was relatively inefficient with either substrate.

Conclusions

NfsA_Nm is a promising new nitroreductase candidate for several diverse biotechnological applications.

     

Biochemical and Immunological Characterization of Nitrate Reductase Deficient nia Mutants of Nicotiana plumbaginifolia

Sixty-five Nicotiana plumbaginifolia mutants affected in the nitrate reductase structural gene (nia mutants) have been analyzed and classified. The properties evaluated were: (a) enzyme-linked immunosorbent assay (two-site ELISA) using a monoclonal antibody as coating reagent and (b) presence of partial catalytic activities, namely nitrate reduction with artificial electron donors (reduced methyl viologen, reduced flavin mononucleotide, or reduced bromphenol blue), and cytochrome c (Cyt c) reduction with NADH. Four classes have been defined: 40 mutants fall within class 1 which includes all mutants that have no protein detectable in ELISA and no partial activities; mutants of classes 2 and 3 exhibit an ELISA-detectable nitrate reductase protein and lack either Cyt c reductase activity (class 2: fourteen mutants) or the terminal nitrate reductase activities (class 3: eight mutants) of the enzyme. Three mutants (class 4) are negative in the ELISA test, lack Cyt c reductase activity, and lack or have a very low level of reduced methyl viologen or reduced flavin mononucleotide-nitrate reductase activities; however, they retain the reduced bromphenol blue nitrate reductase activity. Variations in the degrees of terminal nitrate reductase activities among the mutants indicated that the flavin mononucleotide and methyl viologen-dependent activities were linked while the bromphenol blue-dependent activity was independent of the other two. The putative positions of the lesions in the mutant proteins and the nature of structural domains of nitrate reductase involved in each partial activity are discussed.